Ewing's sarcoma is a childhood/young adult cancer. Unfortunately, many Ewing's sarcoma cancer patients are first diagnosed when the cancer cells are already spreading throughout the body, and these patients have a very poor prognosis. Therefore, identifying novel means to improve the effectiveness of the standard treatment of this cancer, a cocktail of drugs that includes etoposide, would be of immediate benefit. This proposal will provide the basis for novel strategies in the treatment of human cancer. Towards this goal, our work has been to identify the mechanisms that a normal (non-cancer) cell uses to survive exposure to an agent such as etoposide. The first part of our hypothesis is that we can identify novel human etoposide survival mechanisms from a Drosophila RNAi screen. We plan to use this knowledge to determine whether any etoposide survival mechanisms are altered in Ewing's sarcoma cancer cells. The second part of our hypothesis is that we can target etoposide survival mechanisms to restore or improve the effectiveness of etoposide therapy depending on what has been altered in the cancer cell. To achieve these goals, we will build from our currently established methods of identifying survival mechanisms in cells from a model organism; Drosophila cells. Once these genes are determined in Drosophila cells, we can test whether the same genes contribute to survival in mammalian cells. Indeed, we already successfully used this strategy to identify novel alkylation survival genes and pathways in mouse and human cells and demonstrated that pathways critical in Drosophila are also involved in mammalian damage response. The advantage of using fruit fly cells is that they share the same processes to survive exposure to an agent such as etoposide as human cells and we can efficiently, rapidly and cost-effectively ask whether removing any one gene alters the ability of a cell to survive etoposide treatment. This provides us with the ability to identify novel genes/mechanisms involved in cell survival. We will then demonstrate that the survival processes we identify in fruit fly cells are also used in mammalian cells. For this we plan to use human Ewing's sarcoma cells. In Aim 1, we propose to leverage our strategy, first completing the validations for a genomic survival screen to etoposide, identifying survival mechanisms, and then we will determine which of these mechanisms are altered in Ewing's sarcoma cells in a EWS/FLI1 dependent manner and which can be targeted to sensitize these cells to etoposide. In Aim 2, we will directly test the functionality of already identified survival mechanisms (and any identified from Aim 1) in a set of Ewing's sarcoma cancer cells, demonstrating any underlying heterogeneity in these cancers, and whether targeting these different pathways effects etoposide sensitivity in these cells. We are uniquely positioned to carry out the proposed work with the necessary infrastructure, collaborators and technical expertise, as demonstrated by our publications and our preliminary data that supports our hypothesis. Ultimately, our goal is to improve etoposide treatment of Ewing's sarcoma.